CoNS-seq of spike sample (0.1 virus genome copies per T-cell): replicate 3
The extracted DNA/RNA was converted to cDNA using SISPA technique. Purified double stranded DNA from each experimental replicate was subjected to single-plex library preparation and long-read nanopore sequencing. The DNA sequencing library was prepared by following the manufacturer's instructions for the genomic DNA by ligation kit (Oxford Nanopore Technologies, SQK-LSK-110). The library preparation process ensured end-repair, and adapter ligation to all DNA molecules and library cleanup. The following two modifications to the protocol were made: 1) starting DNA masses below 1 g were used for spikes below 10 vgc per cell because purified DNA from these spiked samples typically yielded low DNA mass that were below the detection limit of QubitTM 1X dsDNA high sensitivity kit (Thermo Scientific, Q33230). Libraries for the 10 vgc per cell spikes were detectable by Qubit TM as the average DNA mass was 28 ng; 2) the SFB buffer was used during adapter ligation step to retain all short and long DNA fragments. Singleplex DNA libraries were prepared to minimize the risk of false positives associated with cross-contamination and ensure maximum throughout per flow cell. Libraries from each experimental replicate were loaded onto R9.4.1 flow cells from Oxford Nanopore Technologies (FLO-MIN106, active pore number  800) and sequenced on a GridION device for 36 hours.